Epidemiologic studies have shown that tobacco smoking is a risk factor for colorectal cancer, but the chemicals associated with this risk are unknown. We reported that the heterocyclic aromatic amine, 2-amino-9H-pyrido- [2,3-b]indole (AaC), a rodent carcinogen, is present at high frequency in urine of male subjects of the Shanghai Cohort Study. The urinary concentration of AaC was strongly related to the frequency of cigarette smoking. We hypothesize that the high exposure to AaC through cigarettes, and the propensity of AaC to undergo bioactivation by enzymes expressed in liver and colorectum, provides a biochemical mechanism for the development of colorectal cancer in smokers. Our long-term goal is to determine if AaC in tobacco smoke is causally related to colorectal cancer in smokers, and to elucidate the mechanistic pathways underlying this exposure-cancer relationship. There are three objectives of this application. In Aim 1, the extent of exposure to AaC will be determined, by measuring the levels of AaC in urine and stool of tobacco smokers participating in a smoking cessation study, under a clinical trial setting. In Aim 2, the genotoxicity of AaC in the colorectum of C57BL/6N mice will be determined, by measurement of DNA adducts and aberrant crypt foci, as early biomarkers of neoplasia. This study will provide us with a clearer idea about the carcinogenic potential of AaC in this mouse strain. The demonstration of an organ-specific genotoxic effect of AaC in the colorectum of mice is critical for our hypothesis of a causal role for AaC in tobacco-induced colorectal cancer. In Aim 3, the major pathways of AaC metabolism will be characterized in mice and humans, especially as they relate to DNA adduct formation at the target tissue. These studies will be done with hepatocytes, colorectal cytosols, and in a mouse model where the NADPH- cytochrome P450 reductase gene has been selectively deleted in either the liver or in the small and large intestines. Dosing will be done orally or by inhalation to assess the impact of dose routes on DNA damage in the colon. These studies will reveal the capacities of enzymes across species to bioactivate AaC and the relative roles of P450s in liver and colon to induce DNA damage in the colon. This proposed research is relevant to NIH's mission on public health and highlighted by the Genes, Environment, and Health Initiative, which is designed to address the very questions raised in this application. Our hypothesis of the contribution of an HAA from a non-dietary source to human colorectal cancer risk is highly novel and innovative. The proposed research will fill critical gaps on exposure and the biochemical toxicology of AaC and advance our understanding of the genotoxicity of the most abundant aromatic amine present in mainstream tobacco smoke that is a potential risk factor of colorectal cancer. PUBLIC HEALTH RELEVANCE: 2-Amino-9H-pyrido[2,3-b]indole (AaC) is the most abundant of the heterocyclic amine/aromatic amine carcinogens formed in tobacco smoke and may be a causal agent of colorectal cancer in smokers. Mechanistic studies on AaC genotoxicity in the colon of an animal model and characterization of the metabolic pathways involved in bioactivation and detoxication of AaC in humans are warranted to clarify the potential role of this genotoxicant in human colorectal cancer.